Lignin is a complex phenolic polymer that reinforces the walls of certain cells in higher plants. It is mainly found in the vascular tissues, where its hydrophobicity waterproofs the conducting cells of the xylem and its rigidity strengthens the supporting fiber cells of both the xylem and phloem. It may also play an important role in defense against pathogen attack (Hawkins et al, 1997).

The Arabidopsis mutant sin1 (also known as fah1) is blocked in the conversion of ferulate to 5-hydroxyferulate (ferulate-5-hydroxylase (F5H)); the lignin of the mutant lacks sinapic acid-derived components typical of wild-type lignin, and the mutant lacks sinapyl esters which may play an important role in UV-B resistance (Chapple et al, 1992; Landry et al, 1995). F5H catalyzes the rate-limiting step in syringyl lignin biosynthesis. The ferulate-5-hydroxylase was cloned by T-DNA tagging and was found to be a cytochrome P-450 monooxygenase (Meyer et al, 1996). F5H expression parallels sinapate ester accumulation in developing siliques and seedlings of Arabidopsis (Ruegger et al, 1999).

The brown-midrib (bm3) mutation in maize and sorghum has been shown to be in the caffeic acid 3-O-methyltransferase [EC 2.1.1.68] structural gene responsible for converting caffeate to ferulate (Vignols et al, 1995). This enzyme can utilize either caffeic acid or 5-hydroxyferulic acid as substrates (Tsai et al, 1995), however the alfalfa enzyme has a preference for the latter (Inoue et al, 1998). Suppression of the caffeic O-methyltransferase in the xylem of quaking aspen results in novel phenotypes with mottled or red-brown wood (Tsai et al, 1998). A high amount of coniferyl aldehyde residues in the lignin is the origin of the red-brown coloration (Tsai et al, 1998).

Cinnamate 4-hydroxylase [EC 1.14.13.11] (C4H) [catalyzing the p-hydroxylation of trans-cinnamic acid to produce p-coumaric acid] is the first Cyt P450-dependent monooxygenase of the phenylpropanoid pathway. C4H is widely expressed in various Arabidopsis tissues, particularly in roots and cells undergoing lignification (Bell-Lelong et al, 1997). Expression of C4H is apparently coordinated with both PAL1 and 4CL in response to light and wounding (Mizutani et al, 1997; Batard et al, 1997; Koopmann et al, 1999).

Transgenic plants of tobacco in which the activity of 4-coumarate CoA-ligase [EC 6.2.1.12] (4CL) is very low (due to down-regulation of the endogenous gene(s)) contain a novel lignin in their xylem. The levels of three hydroxycinnamic acids, p-coumaric, ferulic, and sinapic, which were bound to the cell walls, were increased. Some of these hydroxycinnamic acids were linked to cell walls via ester and ether linkages (Kajita et al, 1997). In Arabidopsis antisense suppression of 4CL have a large decrease in guaiacyl (G) to syringyl (S) lignin units. This suggests that there is an unacharacterized metabolic route to sinapyl alcohol which in independent of 4CL (Lee et al, 1997).

At least 3 forms of 4CL are found in poplar; all share similar hydroxycinnamic acid substrate specificities (4-coumarate > ferulate > caffeate), and all are inactive against sinapate (Allina et al, 1998). 4CL is encoded by a gene family in poplar (Allina et al, 1998).

Lignin content and composition is also markedly affected in transgenic tobacco plants down-regulated in expression of PAL and cinnamate 4-hydroxylase (C4H) (Sewalt et al, 1997).

Other genetic modifications have utilized a strategy of down-regulating cinnamyl-alcohol dehydrogenase (CAD) [EC 1.1.1.195] which catalyzes the reduction of hydroxycinnamaldehydes to give hydroxycinnamyl alcohols or "monolignols", the monomeric precursors of lignin (Baucher et al, 1996; Hawkins et al, 1997).

Transcription of genes encoding PAL (phenylalanine ammonia-lyase) [EC 4.3.1.5], COMT (caffeic acid 3-O-methyltransferase) [EC 2.1.1.68] and CCOMT (caffeoyl-CoA 3-O-methyltransferase) [EC 2.1.1.104] is strongly activated in elicitor-treated cell suspension cultures of alfalfa. Whereas PAL activity was increased, COMT and CCOMT activities were unchanged. Elicitor treated cells did not accumulate lignin. This suggests the importance of posttranscriptional events in the control of phenylpropanoid biosynthesis (Mi et al, 1996).

The CCoAOMT (caffeoyl CoA 3-O-methyltransferase) [EC 2.1.1.104] of Vitis vinifera methylates both caffeoyl- and 5-hydroxyferuloyl-CoA (Busam et al, 1997). The alfalfa CCoAOMT has a preference for caffeoyl-CoA (Inoue et al, 1998). CCoAOMT expression is associated with lignification in several dicot species (Ye, 1997; Ye et al, 2001). In cell-suspension cultures of Vitis CCoAOMT activity is induced upon fungal elicitation (Busam et al, 1997). Treatment with fungal elicitor or low concentrations of salicylic acid and some other inducers of the systemic acquired resistance (SAR) response, raise the CCoAOMT transcript abundance (Busam et al, 1997). Tobacco plants expressing antisense CCoAOMT exhibit marked reductions in lignin content and altered lignin composition: guaiacyl lignin was preferentially reduced, resulting in an increase in syringyl/guaiacyl ratio (Zhong et al, 1998).

In Arabidopsis sinapic acid is esterified [on the carboxyl moiety] with glucose (via UDPG:sinapoyltransferase). Sinapoylglucose acts as a sinapate donor in the synthesis of sinapoylcholine via transesterification catalyzed by sinapoylglucose:choline O-sinapoyltransferase [EC 2.3.1.91]. Sinapoylglucose is also transesterified with malate to yield sinapoylmalate catalyzed by sinapoylglucose:malate O-sinapoyltransferase [EC 2.3.1.92] (SMT). A mutant of Arabidopsis (sng1) has been identified which is deficient in the latter activity, and lacks sinapoylmalate accumulation, instead accumulating sinapoylglucose (Lorenzen et al, 1996).

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